Compositions and methods for enhancing metal ion dependent drug therapies

ABSTRACT

Methods and compositions are provided for increasing responsiveness to therapeutic metalloproteases including increasing and/or maximizing responsiveness and preventing botulinum and tetanus toxin resistance due to a functional deficiency of zinc. Also provided are methods for zinc replacement or supplement in lacking individuals comprising the administration of a zinc supplement for a loading period and/or administration of a phytase supplement together with the zinc supplement. Also provided are methods for standardization of botulinum toxin potency assays that provide for greater certainty and margins of safety in the use of products from different manufacturers.

CROSS REFERENCE TO RELATED APPLICATION

This nonprovisional utility application claims priority to related U.S.provisional application No. 61/219,932, filed Jun. 24, 2009, whichapplication is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to compositions and methods used inconjunction with assay and administration of certain pharmaceutics, inparticular, co-factor dependent toxins such as the Clostridialneurotoxins.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with the development of lethal toxins such as botulinumtoxin into useful pharmaceuticals. The disease term “botulism” wasderived from the Latin word for sausage, botulus, on the basis of afatal outbreak in Germany in the 1800s caused by partially cookedsausage. Botulism was later determined to be due to toxins elaborated bythe Clostridium botulinum organism, originally isolated in 1895. C.botulinum is a gram-positive, spore-forming, anaerobic rod commonlyfound on plants, in soil, water, and the intestinal tracts of animals.Botulinum toxin was first purified in 1928 and is among the most toxicsubstances known. Botulinum toxins act to cause paralysis at fourdifferent sites in the body: the neuromuscular junction, autonomicganglia, postganglionic parasympathetic nerve endings, andpostganglionic sympathetic nerve endings that release acetylcholine(Ach).

Several different strains of Clostridium, including C. butyricum, C.baratii, and C. argentinense, have been identified that produceantigenically distinct forms of pharmacologically similar botulinumneurotoxins (abbreviated either as BTX or BoNT). Eight major serotypesof botulinum neurotoxins elaborated by bacteria of the Clostridium genusare now known: A, B, C (C1 and C2), D, E, F, and G. Subtypes of theserotypes may be closely related or more distant. For example BoNT-A1and A2 are 95% homologous while BoNT-A3 and A4 are respectively 81% and88% homologous to BoNT-A1. Botulinum toxin type A (BoNT-A) is the mostpotent toxin, followed by the B and F toxin types. It is these typesthat have been exploited commercially to date.

BoNT-A (Oculinum, now BOTOX® brand, Allergan, Inc.) was FDA approved in1989 for medical treatment of blepharospasm (uncontrolled blinking),strabismus (crossed eyes), Meige's syndrome (bilateral blepharospasmwith concurrent dystonia of the lower face), and hemifacial spasm.Approval to treat cervical dystonia, a neurological movement disordercausing severe neck and shoulder contractions was received in December2000. BOTOX® was approved in the UK for axillary hyperhidrosis(excessive sweating) in 2001 and, in the same year, BOTOX® was approvedin Canada for axillary hyperhidrosis, focal muscle spasticity, andcosmetic treatment of wrinkles at the brow line. In 2002, the U.S. FDAannounced the approval of BOTOX® Cosmetic to temporarily improve theappearance of moderate-to-severe frown lines between the eyebrows(glabellar lines). In July 2004, the FDA approved BOTOX® to treatprimary axillary hyperhidrosis.

BoNT-B has also been developed for clinical use and several products arecurrently commercially available (e.g., MyoBloc® in the United Statesand NeuroBloc® in Europe, Solstice Neurosciences). MyoBloc® was FDAapproved in 2000 for treatment of cervical dystonia. BOTOX® BoNT-A isconsidered to be 50-100 times more potent than Myobloc® BoNT-B for anygiven specific treatment.

A number of different manufacturers worldwide are now producing purifiedBoNT-A under various brand names including Xeomin® (Merz Pharma,Germany), Prosigne® (Lanzhou Inst. For Biol. Prod., China), andNeuronox® (Meditoxin® in Korea, Medy-Tox, Inc., Korea). As with otherbiological products, the biological activity for new batches must bedetermined. This is particularly critical for drugs such as botulinumtoxin, which have considerable toxicity and for which restoration ofnormal neuromusclar activity is prolonged. Botulinum toxin deliveredintramuscularly acts at the neuromuscular junction to cause muscleparalysis by inhibiting the release of Ach from presynaptic motorneurons. The peak of the paralytic effect occurs 4-7 days afterinjection. Approximately 2 months after the administration of botulinumtoxin, the axon begins to expand, and new nerve terminal sprouts emergeand extend towards the muscle surface. These new nerve sproutsre-establish the motor nerve unit and muscle paralysis is reversedtypically within 2-4 months. Overdose is sufficiently critical andrecovery sufficiently prolonged that an antivenin is available in thecase of accidental overdose.

Doses of all commercially available botulinum toxins are expressed interms of units of biologic activity. One unit of botulinum toxincorresponds to the calculated median intraperitoneal lethal dose (LD₅₀)in mice. See Hoffman R O, Helveston E M. “Botulinum in the treatment ofadult motility disorders” Int Opthalmol Clin 26 (1986) 241-50. Althoughcertainly not ideal, the LD₅₀ assay has been retained due to its highsensitivity. The definition of unit applies to all forms of commerciallyavailable toxins, in spite of the fact that a standardized approach topotency testing has not been adopted and inter-manufacturer differencesin mouse LD₅₀ assay protocols have led to considerable variability inthe per unit activity for different products. For example, thebio-equivalence ratio of the Dysport® brand of BoNT-A (IpsenPharmaceuticals, France) to Botox® has been suggested to be between2.5:1 and 4:1 by various studies in patients with blepharospasm andhemifacial spasm. As a consequence, when communicating recommendedbotulinum toxin dosage in particular indications, it is has beenconsidered important to specify the particular brand of toxin being usedbecause the units used in labeling are product specific andnon-interchangeable. Further complications arise due the fact that BoNTis a large and relatively labile molecule. Proper reconstitution of theproduct is important and shelf life is quite limited.

Botulinum toxin is capable of inducing formation of antibodies in humansleading to decreased effect of the toxin over time. See Frueh B R, etal. “Treatment of Bepharospasm with Botulinum Toxin. A preliminaryreport” Arch Opthalmol 102 (1984) 1464. Antibody production is thoughtto be a function of cumulative dose, antigenic load per dose, and timeinterval between injections. Thus, it has been suggested that thesmallest therapeutic dose be given with maximum time interval betweensequential injections. Given the importance of the use of BoNT intreating certain diseases, use of BoNT-F is under investigation inpatients who have become immunologically resistant to serotypes A and B.

Although the botulinum toxins are effective for the majority ofpatients, therapeutic effect can vary widely independent of detectableantibody formation—not only from individual to individual, but also foran individual from treatment to treatment. Further, some populationshave been identified to be more likely to show poor- ornon-responsiveness to the toxins. For example, up to 30% of patientsover the age of 65 may demonstrate decreased botulinum toxin efficacy.The basis of failure of a patient to respond to treatment can bedifficult to discern, given that possible explanations include originalmanufacturer differences in potency, the possibility that the patienthas developed toxin specific antibodies, as well as the possibility thatthe product has degraded or been agitated during reconstitution.However, diminished effect within a particular patient populationsuggests the possibility of another factor as well.

Reports of patient demise from the putative dose-dependent distantspread of very large amounts of botulinum toxin administration havesurfaced in the last few years, so any methods which may decrease thedose of toxin necessary to achieve a desired therapeutic outcome wouldbe beneficial.

From the foregoing, it is apparent that any ability to assure maximalpatient responsiveness in treatment and to provide closerstandardization of potency would represent an important therapeutic andsafety advance.

BRIEF SUMMARY OF THE INVENTION

The present inventor has surprisingly found that administration ofrelatively high amounts of well absorbed forms of zinc prior to,concurrently with, or shortly following therapeutic administration ofbotulinum toxin will enable responsiveness to the toxin in individualswho were previously poorly responsive, and apparently enhance thefunctional potency of botulinum toxins in other individuals as well.Even more remarkable, the present inventor has found that zinc loadingprior to administration of botulinum toxin increased responsiveness invirtually every individual tested. It thus appears clear that the testedindividuals were previously relatively functionally deficient in zincavailable for binding and activation of the botulinum toxin's lightchain (LC) zinc dependent endopeptidase. Because the group ofindividuals who are at increased risk for zinc insufficiency as itrelates to maximum responsiveness to the effects of botulinum toxinpotentially include a large percentage of the patient population (SeeTable 1), the present finding provides several means for maximalpossible zinc dependent responsiveness to botulinum toxin.

TABLE 1 Risk Factors for Zinc Deficiency 1. Diet a. Vitamin supplementsi. Poorly absorbed (inexpensive) inorganic zinc forms ii. Iron iii.Vitamin A iv. Calcium v. Copper b. High phytate intake i. Whole grainbreads and fiber ii. Whole wheat products iii. Cereals iv. Soy v. Oatsvi. Legumes (including peanuts, peanut butter, peas) vii. Beans viii.Corn ix. Rice x. Many pre-prepared foods (preservatives) xi. Mostbeverages (including virtually all carbonated soft drinks) 1. Phosphatecontaining compounds 2. Preservative E391 c. Alcohol consumption i.Decreases Zinc absorption ii. Increases urinary excretion iii. Manywines contain phytates d. Milk-based products containing casein andcalcium e. Many “fiber enriching” foods and supplements f. Vegetarianism(diets low in red meats, poultry, and fish, but high in soy) g. Foodscontaining EDTA preservative 2. Medical conditions a. Infections (viral,bacterial, fungal) b. Burns c. Most chronic illnesses d. Malabsorptioni. Frequent Diarrhea ii. Sprue, etc iii. Constipation with frequentfiber and/or laxative use 3. Pregnancy 4. Age <25 or >65 5. Diuretic Use

In certain aspects disclosed herein, the finding is extended tomaximizing effectiveness of treatment with any compound that isdependent for activity on the availability of metal ions in the tissuebeing treated. In one embodiment, the compound is a therapeuticprotease. In one embodiment, the therapeutic protease is a zincendopeptidase, examples of which include botulinum toxin (BoNT), tetanusneurotoxin (TeNT), and Lyme Disease toxin (LDT) among others.

In one aspect, a method of preparing a subject for therapeutic compoundadministration is provided that includes instructing administration (viaoral consumption or any other desired method) of a metal ion supplementfor a loading period prior to—and in some cases concurrently with orshortly following—the administration of the compound, wherein theinstructed administration of the metal ion supplement is at a sufficientlevel to eliminate a relative functional deficiency of the metal ion asa cause for poor responsiveness to the administered compound. In oneaspect, the compound is a zinc endopeptidase such as a botulinum toxin.The metal ion can be in an inorganic or organic form but is ideallyselected on the basis of sufficient bioavailability according tobioavailability measures known in the art.

In one aspect, a zinc supplement is provided that includes an organiczinc form selected from one or more of a zinc proteinate, a zinc chelateand/or salt with an organic molecule, and a zinc amino acid complex. Thezinc supplement is designed to deliver 10 to 400 mg daily of elementalzinc. In other embodiments, the zinc supplement delivers 30 to 50 mgdaily of elemental zinc.

In further aspects, metal ion absorption is promoted by instructing thepatient to limit consumption of phytates during the loading period. Inone particular embodiment, the subject is further instructed toadminister, e.g., to orally consume, at least one phytase together withthe metal ion supplement.

In one embodiment for maximizing responsiveness to therapeuticbotulinum, tetanus neurotoxin, or lyme toxin and related chimeric orsynthetic toxins, loading of zinc stores prior to, concurrently with, orshortly following treatment with the therapeutic metal ion dependenttoxin is facilitated by the provision of a pre-procedure prep pack thatincludes a quantity of zinc supplements in capsule, powder, liquid,liquid-gel, liposomal suspension, or tablet form, wherein the zincsupplements are sufficient to supply 10 to 400 mg of elemental zincdaily for a loading period prior to treatment of a patient with a toxintogether with instructions directing the patient to take the zincsupplements and reduce intake of phytates. In other aspects of thisembodiment, the pre-procedure prep pack further includes a quantity ofphytase supplements in capsule, powder, liquid, liquid-gel, liposomalsuspension, or tablet form and in sufficient quantity for administrationtogether with the zinc supplements through the loading period.

In another embodiment of the invention, methods of potency testing oftoxins including botulinum toxins are provided that enable a measure ofstandardization across the industry. For these embodiments, oral orparenteral zinc supplementation is administered to test animals for aloading period prior to or simultaneous with toxin potency testing,wherein the zinc supplement has been determined to provide sufficientzinc to maximize responsiveness to administered toxin. Maximumresponsiveness is determined empirically by testing diets or zincsupplementation for maximum responsiveness including by modulating thelevel of zinc supplementation together with maximizing zinc absorption.Zinc absorption can be modulated by controlling dietary phytate levelsas well as by adding phytase to the diet.

In another embodiment of the invention, zinc is supplied to the tissueby topical cream, or local or systemic injection. In other aspects,reconstitution of therapeutic toxins in a zinc-containing solution isprovided for purposes of insuring the presence of adequate zinc formaximal activity of the toxin in the target tissues.

In another embodiment of the invention, zinc and phytase in combinationare used to safely and effectively rapidly increase whole body zinclevels in times of medical need, including, but not limited to,conditions such as wound healing, burn recovery, immune compromise, andmale impotency.

The methods and compositions of the present disclosure provide a remedyfor botulinum toxin relative resistance and therapeutic variability inmany individuals with a resulting greatly improved therapeutic outcome,margin of safety, and reliability of therapeutic effect from treatmentto treatment. Maximization of the potential effectiveness ofadministered therapeutic metallopeptidases allows for administration oflower doses and longer times between readministration in some patientswith a putative attendant lower risk of the development of antibodymediated resistance and a potential decrease in distant spread of thetoxin with undesired effects. The methods for standardization ofbotulinum toxin potency assays herein provided resolve a long standingproblem in the industry and provide for greater certainty and margins ofsafety in the use of products from different manufacturers.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, includingfeatures and advantages, reference is now made to the detaileddescription of the invention along with the accompanying figures:

FIG. 1 depicts the raw data of a trial of supplementation of zinc andzinc plus phytase for four days prior to administration of therapeuticbotulinum toxin.

FIG. 2 depicts the graphic results of the trial of supplementation ofzinc and zinc plus phytase for four days prior to the administration oftherapeutic botulinum toxins with respect to the duration of the toxin'seffect in the patient.

FIG. 3 depicts the graphic results of the trial of supplementation ofzinc and zinc plus phytase for four days prior to administration oftherapeutic botulinum toxins with respect to the patients' subjectiveperceived change in the efficacy of the toxin treatments.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be employed in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

Variability of botulinum toxin action in certain individuals hasheretofore been attributed to different sources or lots of the toxin,improper reconstitution or storage, variations in injection technique,the use of topical anesthetics and/or cooling agents, and antibodymediated resistance. The present inventor considered that botulinumtoxin resistance might have a basis in an insufficiency of availablezinc and has proven this remarkable hypothesis. This finding provides aremedy for botulinum toxin resistance in many individuals with aresulting greatly improved therapeutic outcome and margin of safety. Thefinding is extendable to enhancing responsiveness of individuals toother administered compounds that are active in vivo on the basis ofmetal ion availability including other enzymes such as tetanus and lymetoxins, as well as other conditions wherein a patient may benefit fromincreased whole body zinc levels, such as wound healing, burn recovery,immune compromise, and male impotency. Also provided are methods forstandardization of toxin potency assays that provide for greatercertainty and margins of safety in the use of products from differentmanufacturers.

The term “therapeutic metallopeptidase” as used herein refers to apeptidase administered for therapeutic purposes that requires a metalion for partial or full activity. Examples include the zinc dependentmetalloprotease neurotoxins botulinum toxin and tetanus toxin, both ofwhich inhibit neurotransmitter release.

As used herein the term “Clostridial toxin” refers to isolated zincmetalloprotease neurotoxins natively produced by Clostridial speciesincluding without limitation Clostridium tetani, Clostridium botulinum,Clostridium butyricum and Clostridium beratti, as well as Clostridialneurotoxins made recombinantly including in other microbial genera andspecies. The term “botulinum toxin” refers to botulinum toxin serotypesA, B, C, E, F and G, and subtypes thereof, whether natively produced byClostridial species or made recombinantly. Also included as “botulinumtoxins” are isolated botulinum toxin chains (heavy or light chains),whether isolated from Clostridial species or generated recombinantly.The term “botulinum toxin” also includes novel recombinant chimeras. Forexample, novel recombinant chimeras have been generated between BoNT-Aand E and certain of these forms have enhanced activity over nativeforms. See e.g. Wang, J. et al. “Novel chimeras of botulinum neurotoxinsA and E unveil contributions from the binding, translocation, andprotease domains to their functional characteristics” J. Biol. Chem. 283(2008) 16993-17002.

As used herein, the term “toxin injection” refers to the introduction ofa toxin at a site where therapeutic effect is desired and should alsoencompass the concepts of other toxin delivery systems, including, butnot limited to, topical toxin application, site-directed release ofsystemically administered toxin, or nanosystem delivery vehicles.

The botulinum toxins are natively produced in Clostridium bacteria asrelatively inactive, single polypeptide chains of about 150 kDa weightwith a high degree of amino acid sequence homology among the toxintypes. The single polypeptide is subsequently cleaved into a roughly 100kDa heavy chain (HC) and a 50 kDa light chain (LC). The HC and LC chainsare finally bound together by disulfide bonds to form a heterodimer. Thebotulinum toxin HC is responsible for toxin binding to and translocationinto neurons involved in neuromuscular activity. Once inside the neuron,the toxin LC moiety inhibits neurotransmitter release by proteolyticallycleaving various SNARE (Soluble N-ethylmaleimide-sensitive factorAttachment protein Receptor) proteins involved in the release ofacetylcholine at the neuromuscular junction, thus inhibiting muscularcontraction in response to neuronal stimulation. The LC proteolyticactivity is located at the N-terminus of the LC and functions as azinc-dependent endopeptidase.

At the neuromuscular junction, the mechanism of BoNT action involvesthree steps, binding, internalization, and inhibition ofneurotransmitter release through inactivation of various of the SNAREproteins. Three SNARE proteins, syntaxin 1, SNAP-25 and synaptobrevin(a.k.a. vesicle-associated membrane protein or VAMP) together form themetastable “trans” SNARE complexes that act to dock the synaptic vesicleto the presynaptic membrane.

The SNARE protein targets of the LC endopeptidase differ between certainof the BoNT serotypes. SNAP-25 is synaptosome-associated presynapticmembrane protein required for fusion of neurotransmitter-containingvesicles and is the target for BoNT-A and E. VAMP is the target forBoNTs B, D, F and G. The proteolytic target for BoNT-C1 is the syntaxinmembrane protein.

Tetanus neurotoxin (TeNT) is also produced by bacteria of the genusClostridium and, as with BoNT, is a 150 kDa protein consisting of threedomains. However, after binding to the motoneuron presynaptic membrane,TeNT is internalized and transported retroaxonally to the spinal cord.It is then exported out of the motor neuron cell bodies and internalizedby pre-synaptic nerve terminals, where it acts by cleavingVAMP/synaptobrevin thereby preventing release of the inhibitorytransmitter glycine. Synaptic transmission onto motor neurons ispredominantly inhibitory. As such, prevention of this inhibition resultsin hyperactivity of affected motor neurons and thus results in spasticparalysis by the unregulated contraction (tetany) of the innervatedmuscles. Although not yet clinically available, TeNT has been proposedas potentially useful for treatment of neurological conditions of theCNS.

As previously mentioned, the Clostridial neurotoxins are zinc dependentmetalloproteases. When active, BoNT contains one Zn²⁺ (divalent cationzinc) per molecule. Zinc may be bound to the botulinum toxin LC beforeor after entry into cells but is critical for endopeptidase activity. Invitro studies have shown that spontaneous loss of zinc from toxin sitesis relatively slow, but may be increased with longer time in solution.If the endopeptidase becomes inactive by loss of bound zinc, addition ofexogenous zinc can reactivate the toxin.

Dietary Supplementation to Provide Adequate Levels of Metal IonsRequired for Activity of Metalloproteases. Until the early 1960s, it wasbelieved that zinc deficiency in humans did not occur. Since then, ithas been determined that zinc is an essential trace element and thatzinc deficiency is common. Zinc has been found relatively recently to berequired for the catalytic activity of hundreds of enzymes and thatcells have a tight regulatory apparatus for Zn²⁺ availability, which isindicative of its importance in cellular metabolism. Intracellular zinc(Zn²⁺) is in homeostasis with zinc binding proteins such as themetallothionein-1 (MT-1) family of proteins, which act to control theconcentration of free Zn²⁺ by sequestering and releasing Zn²⁺ whenrequired. However, the dynamics of binding and release of Zn²⁺, thecellular distribution of Zn²⁺, and the homeostatic control of Zn²⁺ arenot well understood.

Clinically relevant zinc deficiency results in growth retardation, lossof appetite, and impaired immune function as well as weight loss,delayed healing of wounds, taste abnormalities, and mental lethargy. Insevere cases, which are largely found in underdeveloped countries, zincdeficiency causes hair loss, diarrhea, delayed sexual maturation,impotence, hypogonadism in males, and eye and skin lesions. While overtzinc deficiency is rare in developed countries, zinc deficiency iscommon in developing countries where cereals constitute a very largepart of the diet.

Based on studies of patients with overt symptoms in developingcountries, it was found that phytates in cereals markedly inhibitabsorption of zinc, iron, and other divalent cations. Phytates, mostlyinositol hexaphosphates and pentaphosphates, are found in abundance inthe whole grain breads and fiber, whole wheat products, cereals, soy,oats, legumes (including peanuts and peas), corn, rice, and many foodproducts touted as rich in fiber. The phosphate groups of thesecarbocyclic polyols form strong and insoluble complexes with zinc andinhibit its absorption.

Subclinical zinc deficiency is difficult to detect. Laboratorymeasurement of zinc nutritional status is difficult due to itsdistribution throughout the body as a component of variousmetalloproteins and other zinc binding proteins, as well as nucleicacids. Measurement of levels of zinc in the body is difficult, and serumor plasma levels of zinc and rates of urinary excretion do not correlatewell with intracellular or tissue zinc levels. See Maret W, Sandstead HH. “Zinc requirements and the risks and benefits of zincsupplementation.” J Trace Elem Med Biol 20 (2006) 3-18. Furthermore,clinical effects of zinc deficiency can be present in the absence ofabnormal laboratory findings. Thus, for clinical assessment of zincdeficiency, risk factors such as inadequate caloric intake, alcoholism,and digestive diseases are considered (Table 1) together with symptomsof zinc deficiency when determining a need for zinc supplementation.

Daily oral intake of zinc is required to maintain a steady state leveldue to a lack of a specialized zinc storage system. Dietary zinc isabsorbed mostly through the small intestine. Increased consumption offoods with a high content of absorbable zinc can remedy overt zincdeficiency. Daily zinc losses have been calculated to be in the order of0.63 mg/day for adult healthy men and 0.44 mg/day for adult healthywomen. U.S. surveys indicate that the average intake of zinc in the U.S.is 14 mg/day in adult men and 9 mg/day is adult women. The RecommendedDaily Allowance for oral zinc is 8 mg/day for adult (non pregnant orlactating) women and 11 mg/day in adult men. In contrast, the DailyValue (DV) for zinc is 15 mg daily. DVs were developed by the U.S. Foodand Drug Administration to help consumers compare the nutrient contentsof products in the context of a total diet. The DV for zinc is 15 mg foradults and children age 4 or older. The Tolerable Upper Intake Level(UL) for zinc has been set by the USDA at 40 mg/day. Gastrointestinaldistress has been reported at doses of 50 to 150 mg/day over prolongedperiods. An emetic dose of zinc is estimated to be 225 to 450 mg ofzinc. Importantly, just 0.26 grams of phytate may inhibit the absorptionof up to 50 mg of oral zinc, and, under medical supervision, patientsmay be given in excess of 200 mg/day of zinc supplementation.

Zinc is highly correlated with the protein content of foods but is moreavailable in animal proteins than in protein rich plant foods. Forexample, although turkey (140 grams contains 4.34 mg of zinc) andchicken (140 grams contains 2.88 mg of zinc) are reasonably good sourcesof zinc, a 120 gram piece of tofu only contains 0.77 mg of zinc. Thecontent of zinc in animal proteins varies widely. For example, 6 mediumoysters provide 77 mg of zinc or over 500% of the RDA. In contrast, 3ounces of beef shanks provide 8.9 mg of zinc.

Many individuals are at risk for subclinical zinc deficiency includingthose who take zinc supplementation but with less well absorbedinorganic forms or in combination with phytates that render the zincunavailable. The present inventor has surprisingly found that prioradministration of relatively high amounts of well absorbed forms of zincwill enable responsiveness to botulinum toxin in individuals who werepreviously poorly responsive. Clearly then, such individuals were likelypreviously functionally deficient in zinc available for binding andactivation of the botulinum toxin LC zinc dependent endopeptidase.However, none of these individuals exhibited overt signs of zincinsufficiency.

Individuals who are at increased risk for zinc insufficiency as itrelates to maximum responsiveness to the effects of botulinum toxinpotentially include a large percentage of the patient population (SeeTable 1). Persons with potentially increased risk for zinc insufficiencyin the present context include those who have diets rich in phytates(salts of phytic acid) which are found in whole grain breads and fiber,whole wheat products, cereals, soy, oats, legumes (including peanuts andpeas), beans, corn, and rice. Also at risk are persons having diets lowin beef, turkey, chicken, or oysters (i.e. vegetarians) or individualswho consume these high zinc foods together with foods and compounds thatrender the zinc unavailable including foods and other compounds high inphytic acid or phosphates.

Many foods and beverages contain phosphates or phytic acid as apreservative. Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate,which has the formula C₆H₆(OPO(OH)₂)₆) is the primary storage form ofphosphorus in many plant tissues including bran and seeds. Phytic acidsare highly reactive and readily form complexes with mineral such asCa²⁺, Fe^(2+/3+), Mg²⁺, Mn²⁺, Cu²⁺ and Zn²⁺, as well as withcarbohydrates and proteins. Due to the ability to chelate divalentcations, phytic acids are among the compounds used to treat hard water,to remove iron and copper from wines, and to inactivate trace-metalcontaminants in animal and vegetable oils. The preservative E391 isphytic acid.

As used herein, a “zinc supplement” means one or more forms of inorganicor organic zinc or combinations thereof for oral administration,including in solid, gel, liposomal and liquid forms. The term inorganiczinc refers to the divalent cation Zn²⁺ and inorganic salts of zinc.Non-limiting examples include zinc chloride (ZnCl₂), tetrabasic zincchloride (Zn₅Cl₂(OH)₈), zinc oxide (ZnO), zinc sulfate (ZnSO₄), andtetrabasic zinc chloride (Zn₅Cl₂(OH)₈). The percentage of elemental zincvaries in these forms. For example, approximately 23% of zinc sulfateconsists of elemental zinc. Thus, 220 mg of zinc sulfate contains 50 mgof elemental zinc.

As used herein, the term “organic zinc” refers to organic zinc complexesas well as zinc proteinates, zinc chelates and salts with organicmolecules and compounds, and zinc amino acid complexes including zinchistidine, zinc methionine (ZnMet), Zinc lysine (ZnLys) complexes, etc.Further non-limiting examples include zinc acetate (Zn(O₂CCH₃)₂), zincascorbate (C₁₂H₁₄ZnO₁₂), zinc aspartate (C₈H₁₀N₂O₈Zn₂H), zinc butyrate(Zn(C₄H₇O₂)), zinc carbonate (ZnCO₃), zinc citrate (Zn₃(C₆H₅O₇)₂), zincgluconate (Zn(C₁₂H₂₂O₁₄)), zinc glycinate (C₄H₈N₂O₄Zn), zinc histidinate(C₁₂H₁₆N₆O₄Zn), zinc ketoglutarate (C₅H₄O₅Zn), zinc lactate(Zn(C₃H₅O₃)₂), zinc malate (C₄H₄O₅Zn), zinc picolinate (C₁₂H₈N₂O₄Zn),zinc propanoate (C₆H₁₀O₄Zn), zinc stearate (C₃₆H₇₀O₄Zn), and zincsuccinate (C₄H₄O₄Zn). As with inorganic zinc salts, the percentage ofelemental zinc varies in these forms. For example with zinc arginate,300 mg supplies 30 mg of elemental zinc.

Certain forms of zinc such as zinc oxide and zinc carbonate areessentially insoluble in aqueous solution and have been generallyconsidered to be poorly absorbed. Single dose administration of 50 mg ofzinc in the readily absorbed forms of zinc sulfate and zinc acetateinduces nausea and vomiting in human adults, while the same dose of zincoxide causes such symptoms in only a small percentage of individuals,presumably on the basis of differences in absorption. See Allen L H.“Zinc and Micronutrient Supplements For Children” Am J CLin Nutr68(Suppl) (1998) 4955. Thus, consideration of adequate dosages ofdietary zinc for purposes of the present invention includesconsiderations of relative rates of absorption.

As used herein, the term “phytase” refers to hydrolase enzymes that areable to catalyze the release of phosphorus from phytic acid and saltsthereof (phytates). Phytases are available from many sources includingplant sources, generally from germinating seeds, and microbes. Phytaseshave been described derived from bacteria such as Bacillus subtilis andPseudomonas species as well as from yeasts and filamentous fungi. Thecloning and expression of the phytase gene from Aspergillus niger hasbeen described among others. See EP0420358. Phytases fall into differenttypes depending on the position specificity of their action phytic acidmolecules. Thus, for example, a 3-phytase (myo-inositol hexaphosphate3-phosphohydrolase, EC 3.1.3.8) first hydrolyses the ester bond at the3-position. A 6-phytase (myoinositol hexaphosphate 6-phosphohydrolase,EC 3.1.3.26) first hydrolyzes the ester bond at the 6-position. Althoughthere are exceptions, plant phytases are generally 6-phytases. Microbialphytases are mainly 3-phytases.

Frequent, long term alcohol consumption is associated with impaired zincabsorption and increased urinary excretion. Other potential risk factorsfor functional zinc deficiency include persons who take supplements thatinterfere with zinc absorption including iron, vitamin A, copper andcalcium supplements; frequently consume milk based products containingcasein; have infections, burns, or have recently had surgery; have anychronic illness; have frequent diarrhea or any malabsorption syndrome;are pregnant and/or are below the age of 25 or over the age of 70.

Given that the risk factors for a functional zinc deficiency in thepresent context are so common, one of the most expedient methods forassuring that sufficient zinc will be available for binding to andactivation of botulinum toxin is to augment zinc stores by oralsupplement administration prior to, concurrent with, or shortlyfollowing administration of the botulinum toxin. In one aspect of theinvention, oral supplements and instructions for their use are provided.In one embodiment, the oral supplements include sufficient zinc toprovide from 10 to 400 mg daily of elemental zinc. The amount of zincadministered varies with the relative absorpsion of the form of zincselected, and well absorbed forms are given in lower doses to avoidemetic effects. Zinc at a level of 10 mg is essentially an RDA level ofzinc. Particularly, in the lower end of the range of 10 to 400 mg ofelemental zinc, co-administration of a phytase at a level of 0.8 to10,000 units is preferably provided for ingestion at the time (i.e.,within a couple of hours) of zinc ingestion in order to prevent phyticacid chelation of the ingested zinc. In one embodiment, phytase isprovided for ingestion together with each zinc ingestion, as well asfurther phytase supplements to be taken with every meal in order tomaximize availability of zinc from all dietary sources. In oneembodiment, a high zinc loading dose is provided, which supplies from30-100 mg elemental zinc daily together with 0.8-10,000 units ofphytase.

Other Co-Preparation Methods for Augmenting the Effects of AdministeredMetalloproteases: The present inventor has demonstrated thatresponsiveness to botulinum toxin can be dramatically improved by aperiod of zinc loading prior to administration of the toxin. Thisfinding indicates that even in persons having a normal western diet andwith no apparent evidence of zinc insufficiency, available zinc islimited at least as it relates to the activity of exogenously addedmetalloproteases that are zinc dependent for activity. In oneembodiment, zinc is administered locally to the tissue that is to betreated with a Clostridial neurotoxin. In one aspect, a zinc solution isinjected into sites for toxin injection at around the time of toxininjection, i.e. before, during and/or after toxin injection or toxintopical administration. In other aspects, zinc is administered in apenetrating cream that is applied topically to sites for toxin injectionat around the time of toxin injection, i.e. before, during and/or aftertoxin injection. In other aspects, zinc is administered through the useof nanodelivery systems. In other aspects, zinc is administered viasublingual administration, nasal spray, eye drops, enemas, “forced air,”transdermally, or rectal administration of any form, including aliposomal suspension or nanodelivery system. In further aspects, a zincsolution may be used to reconstitute the therapeutic toxins. Wherelarger areas or inaccessible areas are to be treated, or to moreaccurately dose the zinc therapy, zinc loading by intravenous orintramuscular injection may be alternatively employed.

Clostridial Neurotoxin Reconstitution Solutions: Removal of Zn²⁺ fromthe light chain (LC) by displacement in vitro with soluble chelatorssuch as ethylenediaminetetraacetate (EDTA) completely abolishesenzymatic activity in cell free or broken cell preparations. However,toxin stripped of its bound zinc can retain activity against intactneuromuscular junctions, presumably because the internalized toxin isable to bind available cytosolic Zn²⁺. See Simpson L L, et al. “The Roleof Zinc Binding in the Biological Activity of Botulinum Toxin” J BiolChem 276 (29) (2001) 27034-41. Although tissue that has been pretreatedwith chelators loses its ability to restore activity to toxin that hasbeen stripped of zinc, addition of a molar excess of Zn²⁺ (20 uM) to thetissue will rapidly restore the activity of zinc depleted toxin. Id.

Studies have shown that zinc does not provide a cryoprotectant orcryopermissive effect in lyophilization of botulinum toxin. See Allerganpatent application Ser. No. 10/976,529, published as US 2005/0214326 andPCT/US2006/038913, published as WO 2007/041664. It was asserted inAllergan U.S. patent application Ser. No. 10/976,529, published as US2005/0214326, that addition of zinc to lyophilization solutions doesprovide some increased potency and enhanced anti-microbial activity.However, further independent studies have shown that the LC of BoNT-Aundergoes autocatalytic fragmentation during purification and storageand that this fragmentation is enhanced by zinc chloride, as well asother chlorides of divalent metals. On this basis, some have recommendedthat the toxin be stored frozen in a low concentration of neutral orhigher pH that is devoid of any metals. Ahmed S A, et al. “FactorsAffecting Autocatalysis of Botulinum A Neurotoxin Light Chain” ProteinJ. 23 (7) (2004) 445-51.

In any event, presently commercially available BoNT pharmaceuticalcompositions lack added Zn²⁺ either prior to lyophilization or as anadditive to a reconstitution solution. For example, BOTOX® brand BoNT-Aavailable from Allergan, Inc. (Irvine, Calif.) consists of purifiedBoNT-A that has been purified from culture by a series of acidprecipitations into a crystalline complex consisting of the active highmolecular weight toxin protein and an associated hemagglutinin protein.The crystalline complex is re-dissolved in a solution containing salineand Human Serum Albumin (HSA) and sterile filtered (0.2 microns) priorto vacuum-drying. BOTOX® is reconstituted with sterile, preservativefree 0.9% sodium chloride (saline) prior to intramuscular injection.Other commercially available BoNT pharmaceutical compositions includethe Dysport® (Ipsen Ltd., Berkshire, U.K.) brand BoNT-A hemagglutinincomplex, which is dried into a powder with HSA and lactose, to bereconstituted with saline before use. MyoBloc® brand BoNT-B is providedas in single use vials of an injectable solution of 5,000 U BotulinumToxin Type B in complex with hemagglutinin and nonhemagglutinin proteinsin 0.05% HSA, 0.01M sodium succinate, and 0.1M sodium chloride at aboutpH 5.6. Thus, activity of the BoNT must rely on whatever zinc remainsbound to the molecule throughout purification and storage as well asZn²⁺ in host tissues for activity.

The results presented herein show that, in most individuals tested, zincloading prior to treatment resulted in improved—sometimes dramaticallyimproved—effects and duration of effects of the treatment. This showsthat there is insufficient Zn²⁺ bound to the toxin molecules for maximalactivity and further shows that the tissues of normal individuals lacksufficient available Zn²⁺ to compensate for lack of sufficient Zn²⁺bound to the molecules. Thus, in one embodiment presented herein, a BoNTreconstitution solution is provided that includes sufficient Zn²⁺ toenable maximum responsiveness of the injected toxin by overcoming anydeficiencies in either toxin bound zinc or tissue resident availablezinc. Given the findings disclosed herein, an ideal level of Zn²⁺ to beprovided in reconstitution solutions for delivery can be determinedempirically. In one embodiment, mouse neurotoxin potency assays areutilized to determine levels and forms of zinc to be added to thedelivery solutions. In one embodiment, the mice are placed on a zincdeficient diet for a clearing period prior to testing of desired levelsof Zn²⁺ to be supplied in delivery formulations. By placing the mice ina state of relative zinc deficiency prior to testing, the contributionof Zn²⁺ present in the delivery solution can be isolated. In oneembodiment of the invention, the BoNT is lyophilized without added Zn²⁺and a reconstitution solution is provided that contains sufficient addedZn²⁺ for maximal activity. In one embodiment, the Zn²⁺ is present in thereconstitution solution at a concentration of from 10-400 μM.

In one embodiment, a reconstitution solution is provided that includesisotonic saline, i.e., approximately 0.9% or 0.155M NaCl in sterileaqueous solution (preserved or non preserved), and further includeselemental Zn²⁺ at a concentration of from about 10 to about 50 μM orabout 0.000065 g to about 0.00032 g/100 ml of elemental zinc. Thisquantity of elemental zinc can be provided, for example, with ZnCl₂ atabout 0.000136 g to about 0.00068 g/100 ml.

Clostridial Neurotoxin Potency Assays: The currently accepted assay ofpotency per vial for botulinum toxin is an intraperitoneal mouse lethaldose (MLD50) assay. Potency can refer to recovered potency of thebotulinum toxin after reconstitution or the potency of the botulinumtoxin prior to lyophilization. One unit (U) of a botulinum toxin isdefined as the amount of botulinum toxin which, upon intraperitonealinjection, kills 50% of a group of female Swiss Weber mice weighing17-22 grams each at the start of the assay. Further specifics of theassay are provided in PCT/US2006/038913, published as WO 2007/041664,and incorporated herein by reference.

The European Pharmacopoeia (Ph Eur) monograph on Botulinum toxin type A(BoNT/A) for injection (01/2005:2113) has supported the use ofalternative non-lethal and ex vivo methods to replace the mouse LD50assay, subject to their validation. (Secardia, D. and Das, R. G.“Alternatives to the LD50 assay for botulinum toxin potency testing:Strategies and progress towards refinement, reduction and replacement”AATEX 14, Special Issue, 581-585. Proc. 6th World Congress onAlternatives & Animal Use in the Life Sciences. Aug. 21-25, 2007,Japan). Proposed alternative potency assays include the mouse flaccidparalysis assay (also known as the mouse abdominal ptosis assay), whichrelates the activity of BoNT/A to the degree of abdominal bulging seenafter the toxin is subcutaneously injected into the left inguinocruralregion of a mouse. The magnitude of the paralysis is dose-dependent. Asonly a sublethal dose of BoNT is injected, the assay is considered to be10-fold more sensitive than the LD50 assay. The paralysis endpoint ismore similar to the clinical use of the toxin because it evaluateslocalized muscle effects, rather than systemic toxicity. The flaccidparalysis assay is more rapid than the lethality test and providesresults in 24 to 48 hours, compared to 72 to 96 hours for a typical LD50assay.

An alternative ex vivo assay has also been proposed and measures theamplitude of a twitch response to electrical stimulation of an excisednerve/muscle preparation. The potency of the toxin determines thedecrease in the amplitude of the twitch response. The usual endpoint ofthe assay is the time until a 50% decrease in amplitude is observed. Theex vivo model can provide results within two hours.

Although the LD₅₀ assay has been retained due to its high sensitivity,the assay is not standardized, and units used in labeling are productspecific and not interchangeable. Absence of standardized testing is asafety issue given the extreme toxicity and slow recovery fromoverdosage. The present inventor has determined that oral administrationof zinc results in considerable improvement in botulinum responsivenessin many patients that do not otherwise exhibit any indicia of zincdeficiency. This remarkable finding has been extended to botulinumpotency assays and provides for maximum responsiveness in the testsystem. In one aspect, a standardized diet is provided to the mice for aperiod of time prior to conducting an intraperitoneal LD₅₀ assay, amouse flaccid paralysis assay or an ex vivo twitch response assay.

A typical research grade rodent diet includes from 14-20% protein and 50to over 70% carbohydrate. The highest percentage ingredients in the dietare high in phytates or otherwise inhibit zinc absorption. These includecasein, corn starch, soybean meal, cellulose and may further includecorn and/or soybean oils. Mineral mixtures are added to the diet butthis is not standardized and can vary by 10 fold in different standarddiets.

In various reported studies, diets containing 25-55 mg of zinc per kg ofchow were considered adequate, while diets containing <1-5 mg of zincper kg were considered deficient. In other reports, zinc values havebeen given in parts per million (ppm) and zinc deficient and adequatediets contained 1.5 and 70-75 ppm of zinc, respectively. It has beennoted that a diet containing 10 mg/kg of zinc may be adequate where theprimary protein source is egg white or casein but that at least 20 mg/kgwould be required where a high phytate protein source such as soybean isused. Mice are relatively resistant to zinc toxicity and have beenreported to not show significant effects from 500 mg Zn/L of water forup to 14 months.

In one embodiment of a method for normalizing Clostridial neurotoxinpotency testing in animals and utilizing animal tissues, animals to beused for potency testing are placed on a defined diet designed tomaximize available zinc for at least one week prior to testing. In oneembodiment the diet is low in phytates and high in zinc. In oneembodiment where a low phytate diet is given, the zinc level is at leasttwo-fold in excess of recommended zinc levels. In one embodiment, aphytase is added to the diet in addition to a zinc level at leasttwo-fold in excess of recommended levels. In one embodiment, the defineddiet is selected by comparison of the potency of the same lot ofClostridial neurotoxin in groups of animals fed different diets for atleast one week prior to the potency analysis. The diet providing thegreatest sensitivity to the effects of the toxin is selected andstandardized as the required defined diet for all future potency tests.

The following examples are included for the sake of completeness ofdisclosure and to illustrate the methods and compositions of the presentinvention as well as to present certain characteristics of thecompositions. In no way are these examples intended to limit the scopeor teaching of this disclosure.

EXAMPLE 1 Reversal of Botulinum Toxin Resistance by Dietary ZincSupplementation

The present inventor reasoned that differences in availableintracellular zinc stores in individuals might explain the lack ofbotulinum responsiveness in some patients. In an initial exploration ofthe hypothesis, zinc supplementation was provided by food sources.Turkey was selected as a dietary source of bioavailable Zn²⁺ on thebasis of safety, low cost, easy accessibility, and general acceptance.The amount of turkey that the patients were directed to consume wascalculated to provide a daily dose of 40 mg, or approximately 2.5 timesthe recommended daily value. A number of these patients were consideredto be poorly responsive to botulinum toxins such that control of theirblepharospasm symptoms had been historically difficult to achieve. Theresults showed that zinc supplementation resulted in remarkablyeffective treatment of blepharospasm using the same botulinum toxinsource and dosage that was ineffective in the same patient in theabsence of supplementation.

The results surprisingly suggested that zinc deficiency sufficient toreduce the efficacy of administered botulinum toxin occurs in asignificant number of patients. Even in those patients without overtresistance, increasing zinc levels by oral administration for a periodof time prior to a planned injection is expected to provide anormalative advantage in all patients. In particular, by administeringzinc supplements to provide intracellular zinc available for binding andactivation of botulinum toxin LC endopeptidase, the highest possibleZn²⁺ dependent response can be obtained. In many patients who have beenpreviously refractory to administered botulinum toxin, lower doses canbe administered with reliable responses. While eating copious amounts ofhigh zinc-containing animal protein for a period of time prior to atreatment with botulinum toxin may be effective in normalizing andaugmenting botulinum toxin responsiveness, this solution isinconvenient, lacks standardization, and, over the long run, isincompatible with the dietary preferences of most individuals.

Thus, in one aspect of the advancement provided herein, oral zincsupplements are administered which supply the desired normalization andaugmentation of zinc stores. In one aspect of the invention, one or moreforms of inorganic and/or organic zinc are formulated in tablet orcapsule form. In one aspect, the dosage of zinc for oral consumption isin a range of about 10 to 400 mg of elemental zinc daily. In otherembodiments, the dosage of zinc is in range of about 30 to 50 mg ofelemental zinc daily. In one embodiment, the dosage of zinc is about 50mg of elemental zinc daily. In one embodiment of the invention, a dosepack is provided to a patient prior to a planned procedure including azinc supplement and instructions for taking the supplement. As providedin the instructions, the patient is directed to begin zincsupplementation several days prior to administration of botulinum toxin.In one embodiment, the patient is instructed to increase zincsupplementation for 3 to 4 days in advance of toxin treatment as well ason the day of treatment. In one aspect, the instructions direct theavoidance of foods high in phytates during the zinc loading period.

EXAMPLE 2 Zinc Supplementation Including Increased Absorption of Zinc byPhytase Administration

Given the role of phytates in inhibiting zinc absorption, in one aspectof the invention, oral zinc absorption is increased by oraladministration of phytases which hydrolyze phytic acids (inositolhexakisphosphate) and improve zinc oral absorption. Phytases arepresently available in the nutriceutical market as dietary supplementsdesigned to increase the absorption of divalent cations such as calcium,magnesium, iron and zinc. These divalent cations form insolublecomplexes with plant phytic acids present in plant foods such as corn,corn by-products, legumes, soybeans and cereal grains, rendering themunavailable. Phytases are used as additives to animal feeds for thepurpose of making phosphates available from phytic acid (inositolhexakisphosphate). Addition of phytase to animal feeds has beendisclosed to be an advantage even in low phytate diets. See e.g.WO9949740, disclosing a low phytate feed composition containing phytasefrom Aspergillus. WO9830681 entitled “Phytase Combinations” disclosesthat a combination of at least two phytases having different positionspecificity is more efficient in releasing phytate phosphorous.

Two types of dietary phytases are presently available, those from cerealand those from microbial sources. Microbial sources include fermentationof fungi such as Aspergillus niger (Finase®, Alko Ltd., Finland) as wellas bacterial fermentation. Phytase activity is determinable by a numberof different assays that measure the amount of enzyme required toliberate a set quantity of inorganic phosphate from phytic acid perminute at defined temperatures and pH. Microbial phytases have a broaderrange of pH activity and have been shown to provide increased ironabsorption in human studies compared with wheat phytase. In one suchstudy, administration of 20,000 phytase units (PU) increased ironabsorption by 14 to 26%. See Sandberg A-S, et al. “Dietary Aspergillusniger Phytase Increase Iron Absorption in Humans” The Journal ofNutrition 126 (1996) 476. Thus, in one aspect a dietary supplement isprovided that includes one or more forms of zinc and at least onemicrobial phytase. Administration of the zinc and the phytase preparethe patient for maximal responsiveness to the later administeredbotulinum toxin.

As mentioned above, zinc and phytase in combination may also be used tosafely and effectively rapidly increase whole body zinc levels forapplications other than improved binding and activation of botulinumtoxin LC endopeptidase. For example, it may be desirable to increasewhole body zinc levels in times of medical need, including, but notlimited to, conditions such as: wound healing, burn recovery, immunecompromise, and male impotency.

In order to further test the apparently positive results of dietary zincsupplementation provided by ingestion of a high zinc food, as well as totest whether addition of phytase would confer a benefit in zincabsorption, a further trial was initiated comparing reactivity tobotulinum toxin in patients after a four day period of testsupplementation with two different supplements compared with a placebo.Each of the test substances and the placebo were variously provided tothe test subjects without identification. Thus, the patients wereblinded to the nature of the supplement. The placebo (P) was a capsuleform of lactulose. The two test preparations were Z₁₀, which was anormal RDA dose of 10 mg zinc in the form of zinc gluconate, andZ_(50P), which was a relatively high zinc loading dose providing 50 mgof zinc in the form of zinc citrate together with 3,000 PHU of phytase(derived from the fungus Aspergillus niger). Two different parameters ofsensitivity to the botulinum toxin were recorded, duration (D) andeffect (E). Duration was expressed as a percentage increase or decreasefrom the patient's “usual” duration of effect because experience showsthat different patients typically experience different effect durations.For some patients, toxin treatments typically last one month, for othersthree months, etc. So, a patient whose toxin usually lasts 90 days andfor whom no change in duration was observed was scored as 0, i.e., nochange from usual duration. A patient who experienced an additional 4weeks (28 days) of toxin effect is calculated to have an increasedduration of 28/90, the ratio of which equals +0.31 and representing aneffect that is 31% longer than usual. Conversely, a negative score suchas −0.25 equals an effect that is 25% shorter than usual. Effect (E) wasexpressed as deviation from the individual patient's usual perception ofcomfort and desired effect. For example, 0=no change from usual effect,−1=slightly less effective than usual, −2=significantly less effectivethan usual, +1=slightly more effective than usual, +2=significantly moreeffective than usual, +3=best effect ever achieved for that patient. Toensure accuracy, patients kept regular logs of perceived effectivenessof the treatment.

The results of a trial of 44 patients are presented in table form inFIG. 1. All of these patients had been treated previously and had aprior recorded history of botulinum toxin responsiveness. Twenty-threeof the patients were considered to be hard-to-treat blepharospasmpatients (BH), meaning they: a.) routinely required >50 units/session ofBotox® brand BoNT-A or another botulinum toxin equivalence; b.)routinely had patient-reported suboptimal results despite maximizedresponse from a customized injection dose and pattern; and c.) reporteda degree of variability in toxin effect from treatment to treatmentdespite unchanging injection dose and pattern. A further seven patientswere blepharospasm patients who consistently responded to BoNT-Atreatment (BC). Four patients were hemifacial spasm (H) patients, whileten patients received cosmetic treatment for wrinkles (C). The threeBoNT types administered are shown on FIG. 1 in the “T” columns as (“B”)BoNT-A (Botox®), (“M”) BoNT-B (Myobloc®), and (“D”) BoNT-A (Dysport®).Patients were treated in a modified randomized, double-blind,placebo-controlled, crossover pilot study.

Study participants were unequally randomized to one of four treatmentgroups by a third party and both the treating physician and patientswere blinded to the pre-injection supplements provided. Twenty-three(52%, group A) underwent pre-injection supplementation with Z_(50P)alone (50 mg zinc citrate plus 3,000 units phytase), six (14%, group B)underwent pre-injection supplementation with both P (lactulose placebo)and Z_(50P) at different times in a cross-over design, four (9%, groupC) underwent pre-injection supplementation with Z₁₀ (10 mg zincgluconate) and Z_(50P) at different times in a cross-over design, and 11(25%, group D) underwent study by pre-injection supplementation with P,Z₁₀, and Z_(50P) at separate times in a triple cross-over design.

Referring now to FIG. 2, a graph 200 illustrating the results of thetrial of supplementation of zinc and zinc plus phytase for four daysprior to the administration of therapeutic botulinum toxins with respectto the duration (D) of the toxin's effect in the patient is shown. Thevertical axis 202 in FIG. 2 represents the percentage change in theduration of the action of the toxin in the patient. The horizontal axis204 shows various bars, each representative of a patient populationreceiving either: a placebo (P) 206; 10 mg of zinc gluconate (Z₁₀) 210;or 50 mg of zinc citrate in addition to phytase (Z_(50P)) 214.Confidence interval bars 208/212/216 represent 95% confidence intervalsfrom the mean. As can be seen in FIGS. 1 and 2, no significant (p>0.05)change from usual, patient-specific treatment duration was seen in theduration (D) of botulinum toxin treatments with pre-injectionsupplementation for patients receiving either placebo (P) 206 or 10 mgof zinc gluconate (Z₁₀) 210, which approximates the RDA for zinc.

Referring now to FIG. 3, a graph 300 illustrating the results of thetrial of supplementation of zinc and zinc plus phytase for four daysprior to the administration of therapeutic botulinum toxins with respectto the patients' subjective perceived change in the efficacy (E) of thetoxin treatments is shown. The vertical axis 302 in FIG. 3 representsthe perceived change in the efficacy (E) of the toxin in the patient.The horizontal axis 304 shows various bars, each representative of apatient population receiving either: a placebo (P) 306; 10 mg of zincgluconate (Z₁₀) 310; or 50 mg of zinc citrate in addition to phytase(Z_(50P)) 314. Confidence interval bars 308/312/316 represent 95%confidence intervals from the mean. As can be seen in FIGS. 1 and 3, nosignificant (p>0.05) changes in effect (E) from patient-specific normalresponsiveness were observed in the patients receiving either placebo(P) 306 or 10 mg of zinc gluconate (Z₁₀) 310, which approximates the RDAfor zinc.

By contrast, in 41 of the 44 (93%) patients given Z_(50P) (zinc citrate50 mg plus 3,000 units phytase supplements) 214 for four days prior tobotulinum injections, a patient-specific average increase in effectduration (D) of 23.6% was observed (p<0.05, See FIGS. 1 and 2). Further,in 38 of 44 patients (86%) given Z_(50P) (zinc citrate 50 mg plus 3,000units phytase supplements) 314 for four days prior to botulinuminjections, the effect (E) of the botulinum toxin treatment was found tobe increased significantly (p<0.05, See FIGS. 1 and 3). Virtually everypatient showed improvement in duration (D), effect (E), or both. In mostcases, both duration (D) and effect (E) were remarkably improved. Infact, in three blepharospasm patients given high zinc loading andphytase (Z_(50P)) who reported decreased beneficial effect, obvioustoxin overdose was observed, making it difficult for them to close theireyes. Thus, even among these three patients, there was an increase intoxin effect. Among other things, the results suggested (and subsequentexperience has shown) that lowered doses of BoNT can be administeredwith reasonable responsiveness in many patients if the patients are zincloaded prior to treatment. By zinc loading prior to administration,maximum responsiveness can be normalized across the patient population,and one important variable in individual responsiveness can beeliminated. Lower effective doses may reduce the subsequent developmentof antibodies that interfere with treatment. This data is felt to behighly reliable, as blepharospasm patients are keenly aware on a dailybasis whether or not they can keep their eyes open to function to read,drive, dress themselves, watch TV, work, shop, etc.

All publications, patents and patent applications cited herein arehereby incorporated by reference as if set forth in their entiretyherein. While this invention has been described with reference toillustrative embodiments, this description is not intended to beconstrued in a limiting sense. Various modifications and combinations ofillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription. It is therefore intended that the appended claims encompasssuch modifications and enhancements.

The invention claimed is:
 1. A method of preparing a subject fortherapeutic metalloprotease administration, comprising: providing asufficient quantity of zinc and a phytase that insures availability of azinc ion required for activity of a therapeutic metalloprotease,administering the zinc and the phytase for a loading period prior to,concurrent with, or shortly following administration of the therapeuticmetalloprotease, wherein the administration of the zinc and the phytaseresults in an availability of the required zinc ion at a sufficientlevel to eliminate a functional deficiency of the zinc ion as a causefor poor or limited responsiveness to the administered therapeuticmetalloprotease.
 2. The method of claim 1, wherein the therapeuticmetalloprotease is a Clostridial neurotoxin.
 3. The method of claim 1,wherein the zinc is delivered in a form that provides elemental zinc inan amount of 10 to 400 mg daily.
 4. The method of claim 3 wherein thezinc comprises an organic zinc form selected from one or more of: zincproteinates, zinc chelates and/or salts with organic molecules, and zincamino acid complexes.
 5. The method of claim 3, wherein the zinc isprovided as an oral supplement that delivers 10 to 400 mg daily ofelemental zinc.
 6. The method of claim 5, wherein the zinc is providedas an oral supplement that delivers 30 to 50 mg daily of elemental zinc.7. The method of claim 1, wherein the administration of the zinc and thephytase is by oral consumption.
 8. The method of claim 1, wherein theadministering of the zinc comprises one or more of the following:topical administration, intravenous administration, intramuscularadministration, oral administration, sublingual administration, nasalspray, eye drops, enemas, “forced air,” transdermally, or rectaladministration of any form, including a liposomal suspension ornanodelivery system.
 9. The method of claim 1, further comprisingdirecting the subject to limit consumption of phytates during theloading period.
 10. The method of claim 7, wherein the zinc and thephytase are provided as oral supplements in separate capsules.
 11. Themethod of claim 1, wherein the phytase is administered at a level of 0.8to 10,000 units daily.
 12. The method of claim 1, wherein the phytase isadministered at a level of 40 to 3,000 units daily.
 13. The method ofclaim 1, wherein the loading period is at least 4 days prior toadministration of the therapeutic metalloprotease.
 14. The method ofclaim 1, wherein the loading period is about 4days or less prior toadministration of the therapeutic metalloprotease.
 15. A method ofpreparing a subject for therapeutic metalloprotease administration,comprising: providing a sufficient quantity of at least one supplement,including a phytase supplement, that insures availability of a metal ionrequired for activity of a therapeutic metalloprotease, administeringthe at least one supplement for a loading period prior to, concurrentwith, or shortly following administration of the therapeuticmetalloprotease, wherein the administration of the at least onesupplement results in an availability of the required metal ion at asufficient level to eliminate a functional deficiency of the metal ionas a cause for poor or limited responsiveness to the administeredtherapeutic metalloprotease.
 16. The method of claim 15, furthercomprising limiting consumption of phytates during the loading period.17. The method of claim 15, wherein the phytase supplement isadministered at a level of 0.8 to 10,000 units daily.
 18. The method ofclaim 15, wherein the phytase supplement is administered at a level of40 to 3,000 units daily.
 19. The method of claim 15, wherein theadministration of the phytase supplement is administered with trace orno additional zinc.
 20. The method of claim 15, wherein theadministration of the at least one supplement further comprises oraladministration of the phytase supplement and co-administration of a zincsupplement.
 21. The method of claim 20, wherein the zinc supplementprovides elemental zinc in an amount of 10 to 400 mg daily.
 22. Themethod of claim 20 wherein the zinc supplement comprises an organic zincform selected from one or more of: zinc proteinates, zinc chelatesand/or salts with organic molecules, and zinc amino acid complexes. 23.The method of claim 20, wherein the phytase supplement provides 40 to3,000 units of phytase daily and the zinc supplement provides 30 to 50mg of elemental zinc daily.
 24. The method of claim 23, wherein thephytase supplement and the zinc supplement are provided to supply about4 days of supplementation prior to administration of the therapeuticmetalloprotease.